1. Field of the Invention
This invention relates to immunochemicalassaying. Immunochemicalassays are proving of immense value in medicine and biology for the assaying of liquid samples, especially for example, body fluid samples such as blood or urine because of the sensitivity and specificity of such assays. The present invention is particularly concerned with assaying for barbituric acid compounds. Accurate assay of these substances is of the utmost value in medicinal diagnosis and control of drug abuse.
In immunoassaying procedures, for a given a target compound, a synthetic antigen is generally first prepared. Heretofore, this has usually been accomplished by coupling the target compound, through a coupling group to a carrier which confers antigenicity to the entire compound. The compound coupled to the carrier is usually known as a hapten and, when coupled, it functions as an antigenic determinant so that the antibodies produced will bind with the hapten. Thus, the antibodies produced should have a distinct and unique character, such that they will bind with only a specific compound or class of compounds. The objective in devising the synthetic hapten-carrier conjugate is to provide a compound which will generate antibodies that are specific to the target compound.
Antibodies are prepared by injecting the synthetic hapten-carrier conjugate into animals and recovering blood serum from the animals after they have had time to generate antibodies. Typical animals are rabbits and goats.
The principal problem is usually that of synthesizing antigens that are capable of producing sufficiently specific antibodies. Biological fluids such as blood and urine frequently contain very closely related compounds and it is common for antibodies to be unable to distinguish the target compound from close relatives, or sometimes even from distant ones. The antibody is then considered to be a poor one and is said to have low specificity and high cross-reactivity.
The assay itself is commonly a competitive binding assay. In a useful embodiment of such an assay, the target compound, which is not necessarily extracted, is allowed to compete with known quantities of a labeled standard to bind with a known quantity of specific antibody. From measurement of the proportion of the labeling in the standard-antibody complex that results, the amount of target compound present can be calculated. Radioactive labeling is particularly convenient. Fluorescence perturbation and electron spin resonance have been used in the art. Normally it is necessary to remove any unreacted labeled standard, before making the determination on the antibody complex, although theoretically, the determination could be made on the removed unreacted portion of the standard.